Molecular mechanism of Na+ -coupled HCO3- transporters: transport of CO3= and CO2

Na耦合HCO3-转运蛋白的分子机制：CO3=和CO2的转运

• 批准号: 10187218
• 负责人: Walter F Boron
• 金额: $ 69.76万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-27 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10187218
• 关键词: 3-Dimensional; Acid-Base Equilibrium; Acids; Active Biological Transport; Address; Anions; Bicarbonates; Binding; Binding Sites; Biological Assay; Carbon; Carbon Dioxide; Carbonates; Cell membrane; Cells; Charge; Chemistry; Computing Methodologies; Consumption; Coupled; Coupling; Data; Dental; Diffuse; Diffusion; Disease; Electrophysiology (science); Epilepsy; Equilibrium; Evolution; Extracellular Fluid; Family; Family member; Feeling suicidal; Gases; Generations; Genes; Genetic Polymorphism; Homeostasis; Human; Hypertension; Illinois; Interdisciplinary Study; Ions; Kidney; Knockout Mice; Laboratories; Lipids; Liquid substance; Maps; Measurement; Membrane; Migraine; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Mus; Mutation; Nephrons; Nucleotides; Oocytes; Organ; Pathogenesis; Pathway interactions; Physiological; Physiology; Play; Process; Proximal Renal Tubular Acidosis; Reaction; Regulation; Research; Role; Single Nucleotide Polymorphism; Site; Speed; Structure; Surface; Techniques; Testing; Thinking; Time; Transport Process; Universities; Work; autism spectrum disorder; base; extracellular; insight; interdisciplinary approach; malignant breast neoplasm; mathematical model; member; molecular dynamics; movement analysis; novel; preservation; simulation; sodium carbonate; tool; voltage clamp

− transporters (NCBTs, members of the SLC4 family) play critical roles in transepithelial HCO−3 Na+-coupled HCO3 transport, whole-body pH regulation, and intracellular pH (pHi) regulation. In this Multi-PI R01, the team will exploit powerful techniques, many developed in their respective laboratories, to elucidate molecular transport mechanisms of NCBTs, which are especially important in the kidney. These tools include surface pH (pHS) measurements, out-of-equilibrium (OOE) CO2/HCO3− solutions, macroscopic mathematical modeling (MMM) of acid-base transport in a single cell, and state-of-the-art molecular dynamics (MD) simulations of interactions between the substrates and the transport molecule, or of CO2 conduction through the NCBTs. In a multidisciplinary approach, the team will answer 2 major questions. Aim 1: Do all NCBTs carry some form of – ion pair—whereas other SLC4 “HCO3− ” transporters actually carry =—arriving or departing as the NaCO3 CO3 HCO3− per se? By monitoring pHS in voltage-clamped oocytes the team will test whether SLC4 family members − . They will address the same question in perfused proximal tubules (PTs) from wild- = ” vs. HCO3 transport “CO3 – binding to the KKMIK region type (WT) and NBCe1-A/D knockout mice. They also test the hypothesis that NaCO3 of TM5 is a rate-limiting step for NBCe1-A transport. Using MD, the team will identify/model outward-facing, occluded, and inward-facing conformational states of NBCe1, NBCn1 and AE1, and identify potential interaction sites –, =, −, Cl−, for NaCO3 Na+, CO3 HCO3 and and use MMM (3D reaction-diffusion simulations) to assess physiological data. Finally, in an iterative process, the team will assess single nucleotide polymorphisms (m- = ” vs. HCO−3 SNPs) as well as other mutations suggested by MD studies, prioritize them, and evaluate for “CO3 transport using physiological assay, interpret using MD and MMM, and suggest new mutations. Aim 2: Do all = ” transport, NCBTs conduct CO2 whereas other SLC4 transporters do not? Having presumably committed to “CO3 evolution faced the challenge of translocating the second carbon atom, ultimately derived from 2×HCO3−. The team will use electrophysiological techniques and a novel neutral buoyancy assay (NBA) to ask whether all NCBTs conduct CO2, whereas other SLC4 members do not. In perfused PTs from WT and NBCe1-A/D knockout mice, they will ask if NBCe1-A conducts CO2 in PTs. The team will use MD to identify potential CO2 pathways through NBCe1, NBCn1, and AE1 as a negative control. MMM will assess the physiological data. Finally, in an iterative process, the team will process m-SNPs and other mutations as outlined in Aim 1, but now for effects on CO2 conduction. The research will reorganize our thinking of NCBT function, providing valuable insight into the pathogenesis of proximal renal tubular acidosis (pRTA) and other maladies associated with NBCe1 (e.g., migraine, ocular and dental abnormalities, suicidal ideation), other NCBTs (e.g., hypertension, breast cancer, epilepsy, autism). The systematic analysis of m-SNPs may provide insights into previously unrecognized “NCBT- opathies.” The work also will have broader impact by elucidating physiological acid-base surface chemistry and = ”. − vs “CO3 for the first time permitting on to distinguish unambiguously among the transport of H+ vs HCO3

−转运蛋白（NCBTs，SLC 4家族成员）在跨上皮HCO−3中发挥关键作用 Na+偶联HCO 3 转运、全身pH调节和细胞内pH（pHi）调节。在此Multi-PI R 01中，团队将 利用强大的技术，许多是在他们各自的实验室开发的，以阐明分子运输 NCBTs的机制，这在肾脏中特别重要。这些工具包括表面pH值（pHS） 测量、CO2/HCO 3 −平衡（OOE）解决方案、宏观数学建模（MMM） 单细胞中的酸碱转运，以及最先进的分子动力学（MD）相互作用模拟 基质和转运分子之间的相互作用，或通过NCBTs的CO2传导。中 通过多学科的方法，团队将回答两个主要问题。目标1：是否所有国家能力建设条约都带有某种形式的 - 离子对，而其他SLC 4“HCO 3-“转运蛋白实际上携带 =-以NaCO 3的形式到达或离开 CO3 HCO 3 −本身？通过监测电压钳位卵母细胞中的pHS，研究小组将测试SLC 4家族成员是否 −。他们将在来自野生型的灌注近端小管（PT）中解决同样的问题。 =“vs. HCO3 运输“CO 3 - 与KKMIK区结合 型（WT）和NBCe 1-A/D敲除小鼠。他们还检验了NaCO 3 TM 5是NBCe 1-A转运的限速步骤。使用MD，团队将识别/建模面向外的， 封闭的，向内面向的构象状态NBCe 1，NBCn 1和AE 1，并确定潜在的相互作用 位点-、=、−、Cl−、 对于NaCO 3 Na+，CO 3 HCO 3和使用MMM（3D反应扩散模拟）来评估 生理数据最后，在一个迭代过程中，研究小组将评估单核苷酸多态性（m- =“vs. HCO−3 SNPs）以及MD研究提出的其他突变，对它们进行优先排序，并评估“CO 3 使用生理学测定进行转运，使用MD和MMM进行解释，并提出新的突变。目标2：做所有事情 =“运输， NCBTs传导CO2，而其他SLC 4转运蛋白不传导？可能致力于“二氧化碳 进化面临着转移第二个碳原子的挑战，最终来自2× HCO 3 −。的 研究小组将使用电生理技术和一种新的中性浮力测定法（NBA）来询问是否所有 NCBTs传导CO2，而其他SLC 4成员不传导。在WT和NBCe 1-A/D敲除的灌注PT中 小鼠，他们会询问NBCe 1-A是否会在PT中传导CO2。该团队将使用MD来确定潜在的CO2途径 通过NBCe 1、NBCn 1和AE 1作为阴性对照。MMM将评估生理数据。最后，在一个 迭代过程中，该团队将处理m-SNP和其他突变，如目标1所述，但现在对 CO2传导。本研究将重新梳理我们对NCBT功能的认识，为认识NCBT的功能提供有价值的见解。 近端肾小管酸中毒（pRTA）和与NBCe 1相关的其它疾病的发病机制（例如， 偏头痛，眼和牙齿异常，自杀意念），其他NCBTs（例如，高血压，乳腺癌， 癫痫、自闭症）。对m-SNP的系统分析可以提供对以前未被识别的“NCBT-1”的深入了解。 病态”这项工作还将通过阐明生理酸碱表面化学和 = ”. - 与“CO 3 这是第一次允许明确区分H+与HCO 3的运输